水稻纹枯病菌细胞壁降解酶组分分析、活性测定及其致病作用

摘要

The kinds of cell wall degrading enzymes (CWDEs) from Rhizoctunia sttlani and their pathogenic effect were analyzed by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme activity measurement, determination of leaky reducing sugar and relative conductivity of leachale from leaf tissues treated with CWDEs. The results of SDS-PAGE of CWDEs revealed that there were at least 10 clear bands of proteins, indicating that there were at least 10 kinds of components with different molecular weight in the mixture of CWDEs. The enzyme activities of seven kinds of common CWDEs' were determined, and the results showed that the activity of polygalacturonase (PG) was the highest, followed by polymethylgalacturonase (PMG). Through comparing the effects of different substrates on the induction of three enzymes, it was found that 1.0% sodium carboxymethyl cellulose (CMC) was the most suitable substrate for the induction of endo-β-1.4-glucanasc (Cx), whereas 1.0% pectin for the induction of PG and PMG. The best enzyme reaction conditions for the three most important CWDEs. i. e. PG, Cx and PMG, were explored from three aspects including temperature, reaction time and pH value. In regarding to reaction temperature, it was found that the best reaction temperature was 30 C forCx, and SO C for PG and PMG. In regarding to reaction time, it was found that 40 min was more suitable for Cx and PMG, whereas 60 min was more suitable for PG. In regarding to pH value, the results indicated that ihe enzyme activities of PG. Cx and PMG were the highest when the pH values reached 5. 0, 9. 0 and 4.0 to 6. 0. respectively. Through the determination of leaky reducing sugar and relative conduclivity of Icachatc from leaf tissues treated with CWDEs. it was obvious thai CWDEs did cause damage to rice leaf tissues and the damage degrees increased with ihe increasing concentrations of CWDEs.%为明确水稻纹枯病菌细胞壁降解酶的种类及其对水稻叶片组织的致病作用,对水稻纹枯病菌的细胞壁降解酶进行了SDS-聚丙烯酰胺垂直板凝胶电泳分析、酶活性测定、叶片浸解后的渗透性还原单糖和相对电导率的测定.水稻纹枯病菌细胞壁降解酶的电泳结果显示,提取的细胞壁降解酶混合物中至少含有10种不同分子量的组分.用3,5-二硝基水杨酸法(DNS法)测定了7种常见细胞璧降解酶的活性,结果显示,以羧甲基纤维素钠(CMC)为诱导底物,内切-B-1,4-葡聚糖酶(Cx)活性最高,其次为多聚半乳糖醛酸酶(PG)和聚果胶甲基半乳糖醛酸酶(polymethylgalacturonase,PMG)；以果胶为诱导底物,PG活性最强,PMG次之.通过比较不同底物对PG、Cx和PMG这三种主要细胞壁降解酶的诱导效果,发现Cx用1.0％CMC的诱导效果最好,PG和PMG用1.0％果胶的诱导效果最好.Cx的最佳反应温度是30℃,PG和PMG的最佳反应温度足50℃；Cx和PMG的反应时间定为40 min较为合适,PG反应时间定为60 min较为合适；当pH值达到5.0时PG酶活性最强,当pH值达到9.0时Cx酶活性最强,pH值为4.0～6.0时,PMG的酶活性达到最强.通过对酶处理后叶片的渗透性还原单糖和相对电导率的测定,发现细胞壁降解酶的确对水稻叶片组织造成了损伤,并且随着酶浓度的增加,损伤程度也逐渐加重.